The development of an antiviral therapy against the AIDS virus, HIV-1, is the main goal of this project. Our strategy is uniquely designed to target HIV expressing cells and to interfere with the replication of HIV in these cells. To accomplish this task, we use a variety of state of the art molecular biological techniques to construct novel defective interfering HIV proviral DNAs with which we plan to target cells that express the HIV by down-regulating the HIV envelope protein on the surface of the HIV infected cell using a chimeric CD4/env protein encoded in the defective HIV genome. In addition, the defective virus encodes ribozymes which cleave HIV genomic RNA and mRNA encoding the envelope protein of HIV. It is our hope that the concerted action of these individual elements will lead to the replication of the defective particle will also spread the interfering genes to uninfected cells, which HIV would normally infect, thereby protecting these cells from a potential high level of HIV production. Toward this goal we have data using a vaccinia virus expression system, which demonstrate that our chimeric CD4/env protein can indeed be expressed in vivo. When transcribed from the same promoter as the env gene, small excess amounts of CD4/env DNA template can inhibit syncytia formation caused by the HIV envelope protein in HeLa T4 cells, suggesting a fine balance of both proteins necessary for cell fusion. We have assembled, cloned and sequenced three proteotype defective interfering HIV proviral DNAs, which when coexpressed with HIV DNA causes an inhibition in the spread of HIV in a population of HeLa T4 cells, as was measured by p24 antigen released from these cells. This interference is caused by the expression of the chimeric CD4/env protein, encoded by the defective interfering virus DNA, which presumably interacts with the HIV envelope protein in the endoplasmic reticulum. Specific deletion of the binding for the env within the CD4 region of the defective proviral DNA construct restores syncytia formation in a coexpression with HIV DNA. To increase the level and selectivity of interference by the defective particle in the future, we have constructed novel multitarget-ribozymes, which all specifically cleave HIV env mRNA at multiple sites in vitro (our latest construct recognizes up to nine different sites). The construction of our fourth to sixth generation prototype defective interfering HIV DNAs are in progress. These DNAs will contain these multitarget-ribozymes as well as packaging sequences for the assembly of the defective interfering genomic RNA into HIV particles. It is our hope that these constructs will combine and be able to carry out all the functions required for antiviral strategy.